1. Study of nontypeable Haemophilus influenza (NTHi) lipooligosaccharide (LOS)-derived oligosaccharide (OS)-protein conjugate vaccine. LOS, an important surface antigens in NTHi, may play a role in the pathogenesis of otitis media with effusion in children. We are attempting to make an LOS-derived OS-protein conjugate as a vaccine candidate to prevent otitis media in an animal model and in humans. A. Selection of vaccine candidate strain. Ten NIHi strains were collected and analyzed for their LOS patterns on SDS-PACE. Whole cells and purified LOSs were injected into mice. Mouse antisera were characterized by bactericidal activities on another four strains. Strain 9274 was selected as a vaccine candidate strain. B. Purification of LOS. Several growth conditions and isolation procedures were examined to obtain a high yield of LOS and outer membrane proteins (OMPs). Bacteria were grown in BJI-hemin-NAD for two days. After centrifugation, outer membrane fragments were purified from the cell pellet. The outer membrane fragments were also purified from the supernatant. The resultant yield is at least a one-fold increase in OMPs and a two-fold increase in LOS over-that from the method of Carlone et al. II. Cell Biology of the Tubotympanum This study is to define cell functions of the eustachian tube and middle ear epithelium in relation to the biological defence mechanism in otitis media using cell culture, organ culture, biochemical, immunological, immunohistochemical, and molecular biological techniques. A. Cell and organ culture techniques will be developed to study the biochemical functions of the epithelial cells and glands in the tubotympanum. B. Purification and characterization for bacterial receptors and LPS receptors from the middle epithelium and oropharyngeal cells using cell culture system. C. Investigate cellular and molecular mechanisms of metaplasia, e.g. keratinization of the ciliated epithelium.